The project will explore the regulatory system that controls the initiation of chromosome separation, a critical event in the life of the cell and an event that often goes awry during tumorigenesis. Following duplication of the chromosomes in S phase of the cell cycle, the resulting sister chromatids are linked together by a protein complex called cohesin. During mitosis, the sister-chromatid pairs are oriented on the bipolar mitotic spindle. At the metaphase-anaphase transition, the cohesin linkage between sister's chromatids is abruptly dissolved by a protease called separase, resulting in synchronous separation of sister chromatids and their movement to opposite poles of the spindle. The proposed studies will explore the control of sister-chromatid separation in the budding yeast Saccharomyces cerevisiae, where much of our knowledge of this process was first uncovered. A key goal of the work will be to identify and characterize the regulatory mechanisms that generate the remarkably robust, switch-like behavior of the anaphase regulatory system. In preliminary studies with yeast cells carrying fluorescent tags on two chromosomes, the synchrony of sister-chromatid separation was found to depend in part on a positive feedback loop that governs activation of separase. These studies also led to the discovery that Chromosome IV consistently separates before Chromosome V, suggesting that chromosomes separate in a specific sequence. The first aim of the proposed studies will be to further characterize synchrony and order in the separation of multiple chromosomes in yeast, and to address the general mechanisms underlying the ordered separation of different chromosomes. The second aim will be to reconstitute the biochemical steps of sister-chromatid separation from purified components, allowing detailed studies of separase activation and cohesin cleavage in vitro. Finally, the third aim will be to use these cellular and biochemical tools to address the mechanisms governing separase activity toward cohesin, with an emphasis on the regulation of cohesin cleavage by protein kinases and phosphatases that control cohesin phosphorylation. The knowledge gained from these studies will provide new insights into the control of chromosome segregation - errors in which often contribute to developmental problems and cancer progression. PUBLIC HEALTH RELEVANCE: When a cell reproduces, the chromosomes are first duplicated and then segregated into a pair of daughter cells. Errors in this process can result in genetic damage or defects in chromosome number, which can accelerate cancer progression or cause developmental defects. The proposed studies focus on the regulatory system that controls the initiation of chromosome separation, with an emphasis on the mechanisms underlying the remarkable robustness and accuracy of this system. These studies will lead to a better understanding of how errors in chromosome segregation can arise in human disease.